Composition for maintaining healthy kidney function

ABSTRACT

The present invention provides a composition comprising prebiotic and probiotic components and is used to reduce elevated levels of nitrogenous waste products and to promote a healthy bowel microenvironment.

INTRODUCTION

This application is a continuation of U.S. Ser. No. 10/936,262 filedSep. 8, 2004, which is a continuation-in-part of U.S. Serial No.PCT/US2002/007554 filed Mar. 13, 2002.

FIELD OF THE INVENTION

The present invention relates to compositions and methods which reducethe concentration of nitrogenous wastes. The compositions may be in theform of a food product, dietary supplement, medical food or drug.

BACKGROUND OF THE INVENTION

One of the main functions of the normal, healthy kidney, besides itsregulatory, endocrine, and metabolic functions, is the disposal of wasteproducts. Any impairment of excretory function can lead to theaccumulation of a variety of nitrogenous waste products including, urea,creatinine and uric acid. High concentrations of waste products in theblood stream can exacerbate renal failure and promote kidney stones.Moreover, nitrogenous solutes in the circulating blood promote osmoticdiffusion into the lumen because of the concentration gradient acrossthe intestinal wall. This diffusion mechanism led to the concept of oralsorbents to augment gut-based clearance of nitrogenous waste products.Sorbents or microbes have demonstrated their ability to remove variouscompounds and nitrogenous wastes within the large bowel.

Urea-specific sorbents such as synthetic polymers and modifiedpolysaccharides have been evaluated for the removal of urea and othernitrogenous wastes via the gut. Other sorbents such as oxidized starch,activated charcoal, and carob flour have also been investigated for thein vivo elimination of uremic toxins with some success. Prakash & Chang((1996) Nature Medicine 2:883-88) demonstrated that microencapsulated,genetically-engineered E. coli DH5 are effective in removing urea andammonia in an in vitro system. The same researchers obtained similarresults in oral administration of E. coli DH5 cells in a uremic ratanimal model. Bliss et al. ((1996) Am. J. Clin. Nutr. 63:392-398) havedemonstrated that supplemental gum arabic fiber increases fecal nitrogenexcretion and lowers urea nitrogen concentration in chronic renalfailure patients consuming a low protein diet. Reinhart et al. ((1998)Rec. Adv. In Canine and Feline Nutr. Iams Nutrition SymposiumProceedings. Vol. II:395-404) found that canine renal patients fed adiet containing a fermentable fiber blend improved clinical end-stagerenal disease status, suggesting that specific nutritional alterationallows repartitioning of nitrogen excretion away from the kidney andinto the feces by colonic fermentation or additional bacterial growth.

Prebiotic components are food ingredients that enhance the actions ofprobiotic components in the digestive tract. In this synergistic orsynbiotic relationship a probiotic component, such as Bifidobacteria,metabolizes undigested carbohydrates, such as dietary fibers,oligosaccharides, etc., to produce short-chain fatty acids such asacetate, propionate and butyrate. These short-chain fatty acids maypromote intestinal cell growth, enhance water and mineral absorption,and prevent yeast, mold, and pathogenic bacterial growth. In addition,probiotic components may antagonize pathogens directly throughproduction of antimicrobial and antibacterial compounds such ascytokines and butyric acid (De Vuyst and Vandamme. Antimicrobialpotential of lactic acid bacteria. In: De Vuyst L, Vandamme E L, eds.Bacteriocins of lactic acid bacteria. Glasgow, United Kingdom: BlackieAcademic and Professional; 1994:91-142; Dodd and Gasson. Bacteriocins oflactic acid bacteria. In: Gasson M J, de Vos W M, eds. Genetics andbiotechnology of lactic acid bacteria. Glasgow, United Kingdom: BlackieAcademic and Professional; 1994:211-51; Kailasapathy and Chin (2000)Immunol. Cell. Biol. 78(1):80-8), reduce gut pH by stimulating thelactic acid-producing microflora (Langhendries et al. (1995) J. Pediatr.Gastroenterol. Nutr. 21:177-81), compete for binding and receptor sitesthat pathogens occupy (Kailasapathy and Chin (2000) Immunol. Cell. Biol.78(1):80-8; Fujiwara et al., (1997) Appl. Environ. Microbiol.63:506-12), improve immune function and stimulate immunomodulatory cells(Isolauri et al. (1991) Pediatrics 88:90-97; Isolauri et al. (1995)Vaccine 13:310-312; Rolfe (2000) J. Nufr. 130(2S):396S-402S), competewith pathogens for available nutrients and other growth factors (Rolfe(2000) J. Nufr. 130(2S):396S-402S), or produce lactase which aids inlactose digestion.

U.S. Pat. No. 4,022,883 discloses a method for alleviating uremicsymptoms in persons suffering from renal failure comprisingadministering orally thereto an effective dosage of a cell mass of anon-pathogenic soil bacteria selected from the group consisting of anurea degrading bacterium, a creatine degrading bacterium, a creatininedegrading bacterium and an uric acid degrading bacterium wherein theurea degrading bacterium is a species of Serratia; the creatininedegrading bacterium is a non-fluorescent Pseudomonas, Rhizobium,Agrobacterium, Corynebacterium ureafaciens, Arthrobacter ureafaciens, E.coli, or Pseudomonas aeruginosa; and a uric acid degrading bacterium isBacillus subtilis, a non-fluorescent Pseudomonas, Bacillus fastidosus,Micrococcus dentrificans, Mycobacterium phlei, Aerobacter aerogenes.

U.S. Pat. No. 4,218,541 teaches a method for converting urea to inocuousproducts. The method involves obtaining a culture of at least onemicroorganism selected from the group of Enterobacter agglomerans, GroupD Streptococcus, Bacilli, and Pseudomonad or mixtures of saidmicroorganisms and adding the culture to a composition containing urea.

U.S. Pat. No. 4,970,153 discloses a method of producing urease and moreparticularly a method of producing acid urease by the cultivation ofLactobacillus fermentum TK 1214. This patent further teaches the use ofsuch acid urease or the decomposition of urea contained in fermentationfood products.

U.S. Pat. No. 5,116,737 teaches a method for growing acid-producingbacterial cultures, such as diary cultures, wherein the culture isselected to contain a urease-producing strain of bacteria and the mediumused for the culturing contains added urea. Urease-producing strains ofStreptococcus thermophilus and Bifidobacterium are also disclosed.

U.S. Pat. No. 5,716,615 discloses a pharmaceutical compositioncontaining several different bacteria including Streptococcusthermophilus, Lactobacilli and Bifidobacteria wherein the bacteria arepresent in the composition at a total concentration of 1×10¹¹ to 1×10¹³per gram. An excipient consisting of maltodextrin, microcrystallinecellulose, maize starch, levulose, lactose or dextrose is furthertaught. Methods of using the pharmaceutical composition are alsodisclosed which include treatment of a gastrointestinal disorder andhypercholesteremia or modulating a host's immune response.

The present invention combines the beneficial effects of prebiotic andprobiotic components into a symbiotic product that effectively reducesnitrogenous wastes.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a compositioncomprising prebiotic and probiotic components. This composition can beadded to a food product, dietary supplement, medical food orpharmaceutical product which, upon ingestion, will promote a healthyintestinal microenvironment and assist in the elimination of nitrogenouswaste products that can build up in concentration in the circulatingblood. Increased concentrations of the wastes are known to exert anegative impact on an individual's physiology and contribute to adecreased sense of well-being and general malaise. Methods for reducingnitrogenous waste products using these products are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effects of nutrient composition and availability ongrowth and urea hydrolysis by KB19, KB4, and KB25. Panel A, B, and Cdepict the growth of KB19, KB4, and KB25, respectively, in variouscombinations of nitrogen and carbon sources in the presence and absenceof urea. Panel D depicts the rate of hydrolysis of urea in 24 hours byKB19, KB4 and KB25 grown in various combinations of nitrogen and carbonsources. Medium 1 was supplemented with peptone and 1% sucrose. Medium 2was supplemented with peptone, ammonium sulfate, and 1% sucrose. Medium3 was supplemented with ammonium sulfate and 1% sucrose. Medium 4 wassupplemented with peptone and 0.1% sucrose. Medium 5 was supplementedwith peptone and a polysaccharide. Medium 6 was supplemented withpeptone, polysaccharide and 0.1% sucrose.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Nitrogenous waste products accumulating in the blood stream havedetrimental affects on health. Removal of nitrogenous wastes bydiverting them into the colon is a viable approach to decrease thenegative impact that waste product accumulation has on an individual'sphysiology. The present invention combines the properties of probioticand prebiotic components into a synbiotic product to effectively reducethe blood concentration of nitrogenous waste products and has the addedbeneficial effect of promoting the growth of desirable intestinalmicroflora.

A probiotic component of the present invention refers to a mono or mixedculture of live or freeze-dried microorganisms which, when applied toman or animal, beneficially affects the host by improving the propertiesof the indigenous microflora, such as bifidobacterium organisms thatmetabolize undigested carbohydrates and are beneficial to an individual.Probiotic components of the present invention are selected for theirability to exert a beneficial effect on the host, survive transitthrough the intestinal tract, to adhere to intestinal epithelial celllining, to produce of anti-microbial substances towards pathogens and/orto stabilize the intestinal microflora. Furthermore, a probioticcomponent should have a good shelf-life. Synbiotic products of thepresent invention generally contain a large number of viable cells atthe time of consumption, and are non-pathogenic and nontoxic. Examplesof probiotic components include, but are not limited to, Bifidobacteriumspp. (e.g., bifidum, longum, infantis), Lactobacillus spp. (e.g.,bulgaricus, acidophilus, lactis, helveticus, casei, plantarum, reuteri,delbrueckii, chamnosus, johnsonii, paracasei), Streptococcus spp. (e.g.,thermophilus, diacetilactis, cremoris, durans, faecalis), Saccharomycesspp. (e.g., pombe, boulardii), Leuconostoc spp. (e.g., citrovorum,dextranicum) and Bacillus sp. (e.g., pasteurii). In one embodiment, theprobiotic component is composed of at least one, at least two, or atleast three microorganisms from the genera Bifidobacterium,Lactobacillus, Streptococcus, Saccharomyces, Leuconostoc and Bacillus.In another embodiment, the probiotic component is composed of at leastone microorganism from the genera Bifidobacterium, Streptococcus orLactobacillus. In a further embodiment, the probiotic component iscomposed of at least two microorganisms from the genera Bifidobacterium,Streptococcus or Lactobacillus. In a particular embodiment, theprobiotic component is composed of Bifidobacterium longum, Streptococcusthermophilus and Lactobacillus acidophilus.

Microorganisms also useful in the invention are those that have theability, either through natural selection or by genetic manipulation, tocatabolize various nitrogenous compounds (e.g., urea, creatinine, uricacid and ammonia) by expressing or overexpressing one or more cognatecatabolic enzymes. Exemplary microorganisms are those having an elevatedlevel of urease or creatininase secretion.

A microorganism exhibiting elevated levels of catabolic enzyme secretioncan be selected or trained by exposing a selected microorganism onincreasing amounts of the metabolite of interest (e.g., urea,creatinine, uric acid and ammonia). For example, it has been found thata standard strain of Streptococcus thermophilus can be trained toexpress elevated levels of urease by sequential passage of the strain onincreasing amounts of urea, e.g., a single colony growing on 0.5% ureais selected and applied to medium containing 1.0% urea, a single colonygrowing on 1.0% urea is selected and applied to medium containing 2.0%urea, etc. Using such a method, a S. thermophilus strain having theability to grow on 5% urea was isolated. This strain proliferated inartificial intestinal fluid (AIF, US Pharmacopeia) in the pH range of5.5 to 7.5, characteristic of the colon environment; used urea as a solenitrogen source; and catabolized urea in the presence of other nitrogensources. It was found that urea hydrolysis was growth- and pH-dependentand that urea concentrations could be reduced by this strain from 300mg/dL to 20 mg/dL within 24 hours at pH 6.3 when inoculated in AIF at aninitial density of 10⁹ cfu/mL. Moreover, this strain survived 3 hours inacidic pH 3.0 with only a one-log loss in cfu and was able to passthrough bile. In addition, this strain did not appear to exhibit anyresistance to eight commonly used antibiotics. Therefore, these dataindicate that a specifically selected or trained bacterial isolate canbe used as a urea-targeted component in a synbiotic product of thepresent invention.

Elevated levels of secretion can also be obtained by overexpressing thegene of interest (e.g., via multiple copies or a promoter driving highlevels of expression) in a prokaryotic microorganism of interest such asBifidobacterium, Lactobacillus, Streptococcus, Leuconostoc or Bacillus,or a eukaryotic microorganism such as Saccharomryces. The gene ofinterest can be under the regulatory control of an inducible orconstitutive promoter. Promoters for use in recombinant prokaryoticexpression vectors are well-established in the art and can include thebeta-lactamase (penicillinase) and lactose promoter systems (Chang etal. (1978) Nature 275:615; Goeddel et al. (1979), Nature 281:544), atryptophan (trp) promoter system (Goeddel et al. (1980) Nucleic AcidsRes. 8:4057; EPO App. Publ. No. 36,776) and the tac promoter (De Boer etal. (1983) Proc. Natl. Acad. Sci. USA 80:21). While these are commonlyused promoters which are commercially available, one of skill in the artcan appreciate that any other suitable microbial promoter can be used aswell. Nucleic acid sequences encoding suitable prokaryotic promotershave been published thereby enabling one of skill in the art to readilyisolate these promoters (e.g., by standard cloning or PCR methodologies)for cloning into plasmid or viral vectors (Siebenlist et al. (1980) Cell20:269). The promoter and Shine-Dalgarno sequence (for prokaryotic hostexpression) are operably-linked to the DNA encoding the gene ofinterest, i.e., they are positioned so as to promote transcription ofthe messenger RNA from the DNA, and subsequently introduced into asuitable host cell.

Eukaryotic microbes such as yeast cultures can also be transformed withsuitable protein-encoding vectors. See e.g., U.S. Pat. No. 4,745,057.Saccharomyces cerevisiae is the most commonly used among lowereukaryotic host microorganisms, although a number of other strains arecommonly available. Yeast vectors can contain an origin of replicationfrom the 2 micron yeast plasmid or an autonomously replicating sequence(ARS), a promoter, DNA encoding the desired protein, sequences forpolyadenylation and transcription termination, and a gene encoding for aselectable marker. An exemplary plasmid is YRp7, (Stinchcomb et al.(1979) Nature 282:39; Kingsman et al. (1979) Gene 7:141; Tschemper etal. (1980) Gene 10:157). This plasmid contains the trp1 gene, whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1(Jones (1977) Genetics 85:12). The presence of the trp1 lesion in theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters formetallothionein, 3-phospho-glycerate kinase (Hitzeman et al. (1980) J.Biol. Chem. 255:2073) or other glycolytic enzymes (Hess et al. (1968) J.Adv. Enzyme Reg. 7:149; Holland et al. (1978) Biochemistry 17:4900),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are commercially available andfurther described in Hitzeman et al., EP 73,657.

As will be understood by those of skill in the art, expression vectorscontaining polynucleotides which encode a degradative enzyme ofinterest, e.g., a urease or creatininase, can be designed to containsignal sequences which direct secretion of enzyme of interest through aprokaryotic or eukaryotic cell membrane. Such signal sequences arewell-established in the art and can be taken from other enzymes/proteinsknown to be secreted into the extracellular environment.

Transforming the microorganisms as defined herein, describes a processby which exogenous DNA is introduced into and changes a recipient cell.It can occur under natural or artificial conditions using variousmethods well-known in the art. Transformation can rely on any knownmethod for the insertion of foreign nucleic acid sequences into aprokaryotic or eukaryotic host cell. The method is selected based on thetype of host cell being transformed and can include, but is not limitedto, viral infection, electroporation, heat shock, lipofection, andparticle bombardment. Such “transformed” cells includestably-transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. This also includes cells which transiently expressthe inserted DNA or RNA for limited periods of time.

As will be appreciated by the skill artisan, a microorganism can also beexposed to a mutagen to cause changes in the genetic structure of theorganism so that it expresses elevated levels of a catabolic enzyme ofinterest.

Transformed or mutagenized strains are subsequently selected for theability to grow in the presence of the metabolite which is degraded bythe catabolic enzyme of interest. By way of example, a straintransformed with nucleic acid sequences encoding a urease is selectedfor high levels of urease secretion by growing said strain on highlevels of urea. Levels of urease secretion can also be detected usingstandard enzymatic assays. As disclosed herein, the strain can besequentially subcultured on increasing levels of urea to further enhanceurease secretion. One embodiment of the present invention provides aurease-secreting strain of Bacillus pasteurii, Streptococcusthermophilus or Saccharomyces pombe. In another embodiment, aurease-secreting strain is at least one of the microorganisms of aprobiotic component composed of at least two or at least threemicroorganisms. In a further embodiment, a urease-secreting strain is atleast two of the microorganisms of a probiotic component composed of atleast three microorganisms.

The probiotics according to the invention can be obtained byfermentation and can be stored after fermentation and before addition tothe synbiotic composition of the present invention for a time and at atemperature that prevents substantial loss of probiotic cfu. Forexample, the probiotic component can be fermented until a finalconcentration of 10⁶ to 5×10¹⁰, or 10⁷ to 10¹⁰, or 10⁸ to 10⁹ cfu per mLof fermented medium is achieved.

When the probiotic component is a mono culture, said mono culture is100% of the probiotic component. When the probiotic component iscomposed of at least two microorganisms, each microorganism can be 10,15, 20, 30, 40, 50, 60, 70, 80, or 90% of the probiotic component,wherein the total of all microorganisms is 100%. An exemplary probioticcomponent is composed of about 10-15% L. acidophilus, about 10-15% B.longum and about 70-80% S. thermophilus (e.g., a ratio of approximately1:1:8, respectively).

As used herein, the probiotic component or urease-secretingmicroorganism is one component or additive to a food product or to anenterically coated tablet, capsule, powder, soft gel, gelcap, or liquid.Accordingly, the probiotic component or urease-secreting microorganismis included at a concentration of 10⁸ cfu/mL, 10⁹ cfu/mL, 10¹⁰ cfu/mL,10¹¹ cfu/mL, or 10¹² cfu/mL when added as a liquid or 10⁸ cfu/g, 10⁹cfu/g, 10¹⁰ cfu/g, 10¹¹ cfu/g, or 10¹² cfu/g when added as afreeze-dried powder. In one embodiment, the probiotic component is about20% to about 70% of the total synbiotic product weight, In particularembodiments, the probiotic component is about 50% of the total synbioticproduct weight.

A prebiotic component of the present invention refers to a non-digestivefood that beneficially affects the host by selectively stimulating thegrowth and/or activity of one or more non-pathogenic bacteria in thecolon. Prebiotic components of the present invention are considered tohave anti-carcinogenic, anti-microbial, hypolipidemic and glucosemodulatory activities. They can also improve mineral absorption andbalance. Furthermore, bacteria belonging to the Bifidobacterium andLactobacillus families are stimulated by the presence of the prebioticcomponent and proliferate. Pharmacokinetically, the prebiotic componentsreach the colon largely intact. An exemplary prebiotic componentincludes, but is not limited to, an oligosaccharide such asfructo-oligosaccharide or inulin, isomaltose oligosaccharide,trans-galacto-oligosaccharide, xylo-oligosaccharide, orsoy-oligosaccharide; a pyrodextrin such as arabinogalactan, lactilol,lactosucrose, or lactulose; or a fiber source such as oat gum, peafiber, apple fiber, pectin, guar gum, psyllium husks, glucomannan orguar gum hydrolysate (BeneFiber, Novartis Pharmaceuticals). In oneembodiment, the prebiotic component is composed of at least one, atleast two, or at least three non-digestive foods (e.g.,oligosaccharides, pyrodextrins or a fiber source). In anotherembodiment, an oligosaccharide is at least one of the non-digestivefoods of a prebiotic component composed of at least two or at leastthree non-digestive foods. In yet another embodiment, a fiber source isat least one of the non-digestive foods of a prebiotic componentcomposed of at least two or at least three non-digestive foods. In afurther embodiment, a fiber source is at least two of the non-digestivefoods of a prebiotic component composed of at least three non-digestivefoods. In a still further embodiment, the prebiotic component iscomposed of at least one of the following non-digestive foods oflactulose, psyllium husks and guar gum hydrolysate. In particularembodiments, the prebiotic component is composed of lactulose, psylliumhusks and guar gum hydrolysate.

When the prebiotic component is a single non-digestive food, saidnon-digestive food is 100% of the prebiotic component. When theprebiotic component is composed of two or more non-digestive foods, eachnon-digestive food can be 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, or 90%of the prebiotic component, wherein the non-digestive food total is100%. An exemplary prebiotic component is composed of about 5-30% byweight of lactulose, 5-30% by weight of psyllium husks, and 5-30% byweight of guar gum hydrolysate.

The amount of prebiotic component added to the probiotic component is100 milligrams per serving, 500 milligrams per serving, 1 gram perserving, 5 grams per serving or 10 grams per serving. In one embodiment,the prebiotic component is not less than 100 milligrams and not morethan 10 grams per serving. In one embodiment, the prebiotic component isabout 20% to about 70% of the total synbiotic product weight, Inparticular embodiments, the prebiotic component is about 50% of thetotal synbiotic product weight. When the synbiotic product furtherincludes addition additives, the percent of the prebiotic and/orprobiotic component can be decreased to accommodate the additionaladditive. In particular embodiments, the percent of the prebioticcomponent is decreased to accommodate the additional additive.

A symbiotic product combining the beneficial properties of probiotic andprebiotic components can include a food product, dietary supplement,comestible medical food or pharmaceutical product. In the context of thepresent invention, “symbiotic” refers to a mixture of at least oneprobiotic and at least one prebiotic components to promote healthenhancing effects (Gibson and Roberfroid (1995) J. Nutr. 125:1401-1412).The ingestion of said synbiotic product reduces the blood concentrationof nitrogenous waste products that accumulate in the circulating bloodstream. These waste products of the present invention can be of anendogenous origin such as normal or abnormal metabolic routes orbacterial putrefaction. Furthermore, the waste products can be of anexogenous origin as in dietary intake of proteins and amino acids.Furthermore, repeated ingestion of the synbiotic product will have ahighly beneficial effect upon the intestinal microflora by localizationand colonization in the large intestine of microbes known to promote ahealthy intestinal microenvironment.

As indicated herein, a synbiotic product of the present invention cantake the form of a food product including, but is not limited to, ahealth bar, health drink, yogurt, dahi, or sachet or an entericallycoated tablet, capsule, powder, soft gel, gelcap, or liquid. In additionto containing at least one, at least two or at least three prebiotic andprobiotic components, the symbiotic product of the present invention canfurther containing various fillers or additives.

Optional additives of the present composition include, withoutlimitation, pharmaceutical excipients such as magnesium stearate, talc,starch, sugars, fats, antioxidants, amino acids, proteins, nucleicacids, electrolytes, vitamins, derivatives thereof or combinationsthereof. In one embodiment, an additive of the symbiotic product iscarob flour, for example, locust bean gum. In another embodiment, anadditive is a mushroom extract from Agaricus bisporus. In particularembodiments, a gel cap contains fillers such as magnesium stearate, talcand starch.

Further, to increase the palatability of a food product containing aprebiotic and probiotic, it may be desirable to add flavors, sweeteningagents, binders or bulking agents.

Flavors which can optionally be added to the present compositions arethose well-known in the pharmaceutical art. Examples include, but arenot limited to, synthetic flavor oils, and/or oils from plants leaves,flowers, fruits and so forth, and combinations thereof are useful.Examples of flavor oils include, but are not limited to, spearmint oil,peppermint oil, cinnamon oil, and oil of wintergreen (methylsalicylate).Also useful are artificial, natural or synthetic fruit flavors such ascitrus oils including lemon, orange, grape, lime, and grapefruit, andfruit essences including apple, strawberry, cherry, pineapple and soforth.

Sweetening agents can be selected from a wide range of materials such aswater-soluble sweetening agents, water-soluble artificial sweeteners,and dipeptide-based sweeteners, including salts thereof and mixturesthereof, without limitation.

Binders can be selected from a wide range of materials such ashydroxypropylmethylcellulose, ethylcellulose, or other suitablecellulose derivatives, povidone, acrylic and methacrylic acidco-polymers, pharmaceutical glaze, gums (e.g., gum tragacanth), milkderivatives (e.g., whey), starches (e.g., corn starch) or gelatin, andderivatives, as well as other conventional binders well-known to personsskilled in the art. Examples of bulking substances include, but are notlimited to, sugar, lactose, gelatin, starch, and silicon dioxide.

When the above-mentioned additives are included in the symbiotic productof the present invention, they are generally less than 15% of the totalsymbiotic product weight. In particular embodiments, they are less than5 to 10% of the total symbiotic product weight.

To facilitate targeting of the synbiotic product of the presentinvention to the gastrointestinal tract, there a number of controlledrelease formulations that are developed preferably for oraladministration. These include, but are not limited to, osmoticpressure-controlled gastrointestinal delivery systems; hydrodynamicpressure-controlled gastrointestinal delivery systems; membranepermeation-controlled gastrointestinal delivery systems, which includemicroporous membrane permeation-controlled gastrointestinal deliverydevices; gastric fluid-resistant intestine targeted controlled-releasegastrointestinal delivery devices; gel diffusion-controlledgastrointestinal delivery systems; and ion-exchange-controlledgastrointestinal delivery systems, which include cationic and anionicdrugs. Additional information regarding controlled release drug deliverysystems can be found in Yie W. Chien, Novel Drug Delivery Systems, 1992(Marcel Dekker, Inc.).

For example, enteric coatings are applied to tablets to prevent therelease of drugs in the stomach either to reduce the risk of unpleasantside effects or to maintain the stability of the drug which mightotherwise be subject to degradation of expose to the gastricenvironment. Most polymers that are used for this purpose are polyacidsthat function by virtue or the fact that their solubility in aqueousmedium is pH-dependent, and they require conditions with a pH higherthan normally encountered in the stomach. One desirable type of oralcontrolled release structure is enteric coating of a solid or liquiddosage form. Enteric coatings promote the compounds remaining physicallyincorporated in the dosage form for a specified period when exposed togastric juice. Yet the enteric coatings are designed to disintegrate inintestinal fluid for ready absorption. Delay of absorption is dependenton the rate of transfer through the gastrointestinal tract, and so therate of gastric emptying is an important factor. Some investigators havereported that a multiple-unit type dosage form, such as granules, may besuperior to a single-unit type.

Typical enteric coating agents include, but are not limited to,hydroxypropylmethylcellulose phthalate, methacryclic acid-methacrylicacid ester copolymer, polyvinyl acetate-phthalate and cellulose acetatephthalate (Hasegawa, (1985) Chem. Pharm. Bull. 33:1615-1619). Variousenteric coating materials can be selected on the basis of testing toachieve an enteric-coated dosage form designed ab initio to have apreferable combination of dissolution time, coating thicknesses anddiametral crushing strength. (Porter et al. (1970) J. Pharm. Pharmacol.22:42p). It is contemplated that the enteric coating can be either foodgrade or pharmaceutical material which is generally used in theproduction of various drug or dietary supplements.

Depending on whether the symbiotic product is to be consumed by an adulthuman, child or animal (e.g., companion animal or livestock), it can beproduced in various sizes and with various ingredients suitable for theintended recipient. For example, while a gel cap size of 0 or 1 may besuitable for humans, a gel cap size of 2, 3, 4, or 5 may be moresuitable for a companion animal.

Further, because the probiotic and prebiotic components of the presentinvention are generally recognized as safe, they can be consumed one,two or three times daily or more.

The present invention also relates to a method for removing nitrogenouswaste products from the blood of an individual with elevated levels ofnitrogen-containing waste products. The method involves administering aneffective amount of a symbiotic product of the present invention so thatthe levels of nitrogenous waste products in the blood are decreased orreduced, desirably to a normal range. For example, normal levels ofcreatinine in the blood are in the range of 0.6-1.2 mg/dL, whereasnormal blood urea nitrogen (BUN) levels range from 7-18 mg/dL and normaluric acid levels in males and females is in the range of 2.1 to 8.5mg/dL and 2.0 to 7.0 mg/dL, respectively. Further, a BUN/creatinineratio of 5-35 is indicative of normal levels of nitrogenous wasteproducts in the blood. As one of skill in the art can appreciate, meansfor determining the levels of nitrogenous wastes are well-known to theskilled laboratory clinician.

As symbiotic products of the present invention containing a S.thermophilus strain that secretes elevated levels of urease have beenshown to reduce the levels of nitrogenous waste products in the blood ofa stable model of chronic renal failure, these compositions are usefulin the a method for treating or preventing renal failure. The methodinvolves administering a symbiotic product of the present inventioncontaining a S. thermophilus strain that secretes elevated levels ofurease to a subject having or at risk of having renal failure. Subjectshaving or at risk of having renal failure include those with diabeticnephropathy, hypertensive nephrosclerosis, glomerulonephritis,interstitial nephritis, or polycystic kidney disease wherein nephronfunction is impaired thereby decreasing glomerular filtration rate.Desirably, an effective amount of a synbiotic product for treating renalfailure is an amount sufficient to effect beneficial or desired results,including clinical results, and, as such, an effective amount of asynbiotic product is one which results in the alleviation oramelioration of one or more symptoms associated with renal failure(e.g., a build up of uremic solutes), diminishment of extent of disease,stabilized (i.e., not worsening) state of disease by supporting healthybowel function, delay or slowing of disease progression, or ameliorationor palliation of the disease state. Treatment can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Typical signs or symptoms associated with renal failure

It is further contemplated that compositions of the present inventioncontaining microorganisms secreting elevated levels of urease may havefurther utility in the prevention or treatment of gout and kidneystones.

Example 1 Yogurt Food Product

A yogurt food product can be prepared from one gallon of commerciallyavailable whole, homogenized, pasteurized milk which is heated toboiling and quickly allowed to cool to approximately 45° C. To this isadded approximately one ounce of yogurt starter culture containinglactic acid bacteria of the genus Lactobacillus, Streptococcus, Bacillusor Bifidobacteria. The mixture is mixed well and allowed to ferment at37° C. for 10 to 12 hours. One or more prebiotics, whole fruitadditives, flavoring, sweetening agents, binders, or other additives canbe combined and added to the yogurt to obtain a product of desiredconsistency or to suit the palette of the prospective consumer. In oneembodiment of the present invention, a food product comprises componentsto meet the special dietary needs of individuals with renalinsufficiency.

Example 2 Health Bar

Health bars can be prepared by combining various excipients, such asbinders, additives, flavorings, colorants and the like, along with theprebiotic (e.g., Lactobacillus, Streptococcus, Bacillus orBifidobacteria) and probiotic components, and mixing to a plastic massconsistency. The mass is then either extruded or molded to form “candybar” shapes that are then dried or allowed to solidify to form the finalproduct.

Example 3 Medical Food

A medical food can be prepared by combining rolled oats, dehydratedapples, honey, inulin, carob flour, cinnamon, sugar, vanilla extract,and lyophilized cultures of L. acidophilus and or L. fermentum, aBifidobacteria, and Streptococcus thermophilus (10⁸-10¹⁰ cfu each).These ingredients are mixed in appropriate proportions with a prebioticand formed into a rectangular bar approximately 12.5 to 15 centimetersin length, 3 to 4 centimeters in width and 1 centimeter in height andplaced into a sterile vacuum oven for 12 to 24 hours to obtain an ediblefood product of the desired consistency.

Example 4 Dietary Supplement

A dietary supplement of the present invention can be prepared bycombining lactic acid bacteria such as Lactobacillus acidophilus (10 to15%) and Bifidobacteria longum (10-15%), and a urease-secretingmicroorganism such as Streptococcus thermophilus (70-80%), asepticallyfreeze-drying the bacteria and combining the processed bulk bacteria(e.g., about 50% of the total synbiotic product weight) with theprebiotic component (e.g., 5-50% by weight of each of lactulose,psyllium husks, and BENEFIBER, wherein the final prebiotic component isabout 45% of the total synbiotic product weight). Fillers such asmagnesium stearate, talc and starch (e.g., about 5% of the totalsymbiotic product weight) are added to the prebiotic and probioticcomponents and enterically coated gel caps are produced according to themethod of Kim et al ((1988) J. Indust. Microbiol. 3:253-257).Approximately 10 to 25 billion CFU of the freeze-dried microorganism iscontained in each capsule (i.e., approximately 30 to 75 billion CFUmicroorganisms per gram) that is enterically coated withhydroxy-propylmethyl cellulose phthalate by spraying over a fluidizedbed of capsules. The resulting dietary supplement has a low surfacearea, is relatively non-porous and can protect the contents therein fromlow pH as is found in the gastric environment for several hours, andwill release the contents into the bowel wherein the pH is relativelyneutral or slightly alkaline. Advantageously, approximately 90-95% ofthe microorganisms can survive to be released into the gastricenvironment.

Example 5 Pharmaceutical Product

A pharmaceutical product for persons suffering from renal insufficiencycan be prepared by aseptically freeze-drying one or more probioticmicroorganisms (e.g., a Lactobacillus, a Bifidobacterium, and aStreptococcus). The processed bulk microorganisms are then combined withthe probiotic component and prepared as enterically coated capsulesaccording to the method of Kim et al ((1988) supra) or tablets, powders,soft gels, gelcaps, or liquids according to standard methods. Forexample, the prebiotic and probiotic components in each capsule areenterically coated with hydroxy-propylmethyl cellulose phthalate byspraying over a fluidized bed of capsules. The resulting pharmaceuticalproduct has a low surface area, is relatively non-porous and can protectthe contents therein from low pH as is found in the gastric environmentfor several hours, and will release the contents into the bowel whereinthe pH is relatively neutral or slightly alkaline.

Example 6 ⅚^(th) Nephrectomized Minipig Model of Chronic Uremia

The effects of a symbiotic composition of the present invention in theform of a gel cap product or formulation admixed with the pig chow weretested using an established model of chronic uremia (mild to moderate)in the Gottingen strain of miniature swine (Willis, et al. (1997) J.Endourol. 11(1):27-32). At sexual maturity (3 months of age) these pigsweigh 8-11 kg, and are about half the size of all other strains ofminipigs. The pigs grow slowly and double their body weight in about 6months.

In this uremic model, ⅚ of the renal mass is removed through bilateralflank incisions (McKenna, et al. (1992) J. Urol. 148(2 Pt 2):756-9). Onekidney is entirely removed, and both poles of the contra lateral kidneyare removed with the aid of electro-cautery (for scoring the renalcapsule and about 2 mm of parenchyma), surgical staples, and Gel foamsutured over the exposed parenchyma of the remnant. The contra lateralkidney is removed during the same surgical procedure by gross dissectionand ligation and section of the renal artery and vein and ureter. Thepigs used in this analysis characteristically experienced a large, acuteelevation of creatinine and Blood Urea Nitrogen (BUN) concentrations inplasma; followed by a decline over 1-2 weeks to values that stabilizedwell above baseline. These pigs maintained their appetite and stableuremia for 3-6 months, maintained or gained body weight, and behaved nodifferently than normal control pigs. Hematocrits declined slowly as theuremia progressed and hemoglobin concentrations also declined over time.After the surgery, the pigs were allowed to recover for 3-4 weeks priorto administering the synbiotic compositions.

Using this model, several different blinded symbiotic formulations inthe form of gel caps or admixed with the pig chow were tested asgut-based therapy for uremia. Formulations administered with the pigchow did not demonstrate any significant differences either in BUN orcreatinine values. However, ⅚^(th) nephrectomized mini pigs (n=6) givena formulation containing four microbial strains (L. acidophilus, L.bulgaricus, B. longum and S. thermophilus; 10×10⁹ CFU/gel cap) exhibitedcontinued body weight gains of approximately 31% and decreased BUN andcreatinine levels of approximately 13% each. Similarly, two ⅚^(th)nephrectomized mini pigs given a formulation containing three microbialstrains (L. acidophilus, B. longum and S. thermophilus; 10×10¹⁰ CFU/gelcap) also showed a decrease in BUN (average 21%) and creatinine (average29%) levels although the body weight of one mini pig increased (6%) andthe other decreased (20%). Additional formulations were tested in theform of gel caps or admixed with food and in general the resultsindicated that oral treatment with a probiotic formulation effectivelyand significantly reduced plasma creatinine concentrations by 22.5±8.5%in a stable porcine model of chronic renal failure. Small sample sizemay have prevented detection of corresponding reductions in BUN andelevation of hematocrit.

Example 7 Animal Dosing of Synbiotic Gel Cap

Table 1 provides a suitable approximate dosage of a gel cap (e.g., asdescribed in Example 4) for administration to an animal such as acompanion animal.

TABLE 1 Weight pounds (Kg) Morning Dose Evening Dose Less than 2.2 lbs(<1 Kg) 1 0 2.2-4.4 lbs (1-2 Kg) 1 1 4.4-8.8 lbs (2-4 Kg) 2 1 8.8-17.6lbs (4-8 Kg) 2 1-2 17.6-35.2 lbs (8-16 Kg) 2 2 35.2-70.4 lbs (16-32 Kg)2-3 2-3 More than 70.4 lbs (>32 Kg) 3 3

Example 8 Urease-Secreting Strains of Streptococcus thermophilus

This example discloses the isolation and selection of a high levelurease-secreting strain of Streptococcus thermophilus. Three isolates ofgram-positive, lactic acid-producing non-pathogenic cocci ofStreptococcus thermophilus were isolated from various sources anddesignated KB4, KB19, and KB25. KB4 was isolated from a probioticproduct, KB19 was isolated from a commercial yogurt product and KB25from Dahi yogurt (from India).

Growth rates and urea hydrolysis of these bacteria in the intestinal pHrange (pH 5.5, 6.3 and 7.5) were determined by transferringexponentially growing cultures of KB19, KB4 and KB25 into modifiedArtificial Intestinal Fluid M2 (AIF, US Pharmacopeia) supplemented with100 mg/dL filter-sterilized urea, 100 μM NiCl₂, 10% MRS broth, dextroseto final concentration of 1%, and 0.3% yeast extract, wherein theinitial cell density was 10⁹ cfu/mL. Pancreatin was omitted from therecipe to allow the evaluation of bacterial growth by direct OD600 nmmeasurement. Urea concentration in the supernatants (% of control) andgrowth (OD600 nm) were measured every 4 hours.

Concentration of urea in the supernatants of bacterial cultures wasmeasured using the protocol and standards supplied with the Blood UreaNitrogen Reagent Kit (535, Sigma, St. Louis, Mo.). Urea hydrolysis wasmonitored by comparing urea-nitrogen concentrations in bacterialsupernatants to appropriate control medium incubated in the sameconditions and expressed as percent of control. Four to nine independentexperiments were conducted and Student t-test was used for statisticalanalysis.

Under similar assay conditions, exponentially growing cultures of KB19,KB4 and KB25 were inoculated into AIF M2, pH 6.3, supplemented with 100mg/dL urea and with or without 100 μM NiCl₂ at initial cell density of10⁹ cfu/mL to determine whether the growth and rates of urea hydrolysisby these strains was dependent on the additional Ni++. Ureaconcentration in the supernatants as a % of control and growth (OD600nm) were measured every 4 hours. Four to nine independent experimentswere conducted and Student t-test was used for statistical analysis.

Similarly, it was determined whether the growth and rate of ureahydrolysis of these strains was dependent on urea concentration. Undersimilar growth conditions exponentially growing cultures of KB19, KB4and KB25 were inoculated into AIF M2, pH 6.3, supplemented with 100 μMNiCl2 and 100, 200, or 300 mg/dL urea. Urea concentration in thesupernatants as a % of the control and growth (OD600 nm) were measuredevery 4 hours. Four to nine independent experiments were conducted andStudent t-test was used for statistical analysis.

The survivability of these KB19, KB4 and KB25 was determined inartificial gastric juice in the presence and absence of urea anddextrose. The average loss in viable cell count after exposure toartificial gastric juice (logs cfu/mL) is shown in Table 2.

TABLE 2 PH/Additive KB19 KB4 KB25 1.4 7 7 7 2.0 7 7 7 2.5 3 4 4 2.5/Urea3 4 4 2.5/Dextrose 3 3 3 2.5/Urea + Dextrose 3 3 3 3.0 2 2 3 3.0/Urea 23 3 3.0/Dextrose 1 2 3 3.0/Urea + Dextrose 1 2 2

Initial cell density was 107 cfu/mL. Urea and dextrose concentrationswere 10 mg/mL and 1%, respectively.

Further it was determined whether the nutrient composition andavailability had an affect on growth and urea hydrolysis by KB19, KB4,and KB25. Each strain was grown for 24 hours at 37° C. and pH 6.0 in thepresence or absence of 100 mg/dL of urea and combinations of nitrogenand carbon sources. The results of this analysis are shown in FIG. 1.

Further analysis of S. thermophilus KB19 indicated that this straincould survive a 3 hour exposure to gastric juice, pH 3.0, followed by a3 hour exposure to 0.3% oxgal, pH 6.0, with only 1 log loss inviability. Remaining viable cells were able to proliferate in AIF M2, pH6.0, supplemented with 230 mg/dL urea and completely hydrolyzed the ureawithin less than 18 hours (n=4). All test solutions were supplementedwith 230 mg/dL urea and 1% dextrose.

Collectively, these analyses indicated that all three strains studiedproliferated in the fed state AIF medium in the pH range from 5.5 to7.5, characteristic of colon environment; they could all use urea as asole nitrogen source; and they each catabolized urea in the presence ofother nitrogen sources. Urea hydrolysis was growth and pH dependent.Under the conditions tested, the rate of urea hydrolysis wasstrain-dependent in tests of pH stability: KB19=KB25>KB4; Nirequirement: KB25>KB19>KB4; urea hydrolysis for over 300 mg/dL:KB19=KB25>KB4; and specific nutrients: KB19>KB25>KB4. Further, there wasstrain-dependent results relating to survivability, wherein in tests ofgastric juice stability: KB19>KB4>KB25; and bile stability:KB19>KB4>KB25.

In view of the desirable traits exhibited by S. thermophilus KB19 it wasfurther determined, using a disc-diffusion test, that K19 was sensitiveto Spectinomycin (100 μg), Kanamycin (30 μg), Chloramphenicol (30 μg),Spectinomycin (100 μg), Penicillin (10 IU), Carbenicillin (100 μg),Doxycycline (30 μg), and neomycin (30 μg).

To further enhance the levels of urease secreted by strain K19, thisstrain was trained on increasing levels of urea as described herein.

1. A composition comprising Lactobacillus acidophilus, Streptococcusthermophilus, Bifidobacterium longum, and psyllium husks, wherein saidcomposition is enterically coated.
 2. The composition of claim 1,wherein the bacteria comprise 20% to 70% of the total weight of thecomposition.
 3. The composition of claim 1, wherein the psyllium huskscomprise 20% to 70% of the total weight of the composition.
 4. A methodof removing nitrogenous waste products comprising administering aneffective amount of a composition of claim
 1. 5. A method of treatingrenal failure comprising administering an effective amount of acomposition of claim 1.